Blockade of sodium channels by phenol derivatives

ABSTRACT

The invention relates to a pharmaceutical composition comprising at least one phenol derivative represented by formula (I) 
                         
wherein
         R 1  represents a hydrogen atom, a halogen atom or a hydrocarbon group containing up to 12 carbon atoms;   R 2  represents a hydrogen atom or a C 1 -C 7  alkyl group; wherein   R 1  and R 2  may optionally form a carbocyclic 5- or 6-membered ring;   R 3  represents a hydrogen atom, a halogen atom or a C 1 -C 7 alkyl group;   R 4  represents a hydrogen atom, a C 1 -C 7  alkyl group or a halogen atom;   R 5  represents hydrogen atom, a halogen atom or a C 1 -C 7  alkyl group; and   R 6  represents a hydrogen atom, a C 1 -C 7  alkyl group or a C 2  or C 3  alkenyl group,   under the proviso that R 2  and R 4  can only both represent a hydrogen atom if both R 1  and R 5  represent a C 1 -C 7  alkyl group and R 3  represents a halogen atom.       
     The composition is particularly useful for the blockage of sodium channels and/or influencing the kinetics of sodium channels and thus can be used as local anesthetic, antidysrhythmic, anticonvulsant/antiepilepticand spasmolytic.

FIELD OF THE INVENTION

The invention relates to a pharmaceutical composition comprising atleast one phenol derivative which is particularly useful for theblockade of sodium channels and/or influencing the kinetics of sodiumchannels. In the pharmaceutical field the composition can be used aslocal anesthetic, antidysrhythmic, anticonvulsive and spasmolytic.

BACKGROUND OF THE INVENTION

Phenol derivatives have a wide variety of clinical uses. Some are usedas bacteriostatic stabilizers in parenteral drug formulations. We haverecently shown that 4-chloro-3-methylphenol (4-chloro-m-cresol) andbenzylalcohol both block muscle sodium channels in a voltage-dependentmanner (Haeseler et al., Br. J. Pharmacol., 128 (1999) 1259-67; Haeseleret al., Br. J. Pharmacol., 130 (2000) 1321-1330). For the anestheticpropofol (2,6-di-isopropylphenol), various voltage-operated (Rehberg &Duch, Anaesthiology, 91 (1999) 512-20; Saint, Br. J. Pharmacol., 124(1998) 655-662) and ligand-gated (Sanna et al., Br. J. Pharmacol., 126(1999) 1444-54) channels, mainly in the central nervous system, havebeen identified as possible targets. Integrated into larger molecules,phenol derivatives with single substituents form the aromatic tail ofmost local anesthetics and class Ib antidysrhythmic drugs. The effect ofthe local anesthetic lidocaine(2-diethylamino-2′,6′-dimethylacetanilide) on voltage-operated sodiumchannels in different excitable tissues has been extensively studied.Lidocaine-induced sodium channel blockade is characterized by a higheraffinity of the drug for fast and slow inactivated channels comparedwith the resting state, and by prolonged recovery from inactivation,introducing a second, slow component representing drug dissociation frominactivated channels (Balser et al., J. Gen. Physiol., 107 (1996)643-658; Bean et al., J. Gen. Physiol., 81 (1983) 613-642; Fan et al.,J. Physiol., 81 (1996) 275-286; Scheuer, J. Gen. Physiol., 113 (1999)3-6; Vedantham & Cannon, J. Gen. Physiol., 113 (1999) 7-16). Severalstudies have addressed the structural requirements for pharmacologicaleffects (Ehring et al., J. Pharmacol. Exp. Therapeut., 244 (1988)479-92; Sheldon et al., Mol. Pharmacol., 39 (1991) 609-614), but havenot provided clues about which parts of the lidocaine molecule areresponsible for its state-dependent interaction with the sodium channel.The approach of dissecting the lidocaine molecule into phenol anddiethylamide (Zamponi & French, Biophys. J. 65 (1993) 2335-2347) did nottake into account the fact that the aromatic group of the parentcompound is a methylated phenol derivative. Although phenol blockmimicked slow block of cardiac sodium channels seen with lidocaine,blocking potency was an order of magnitude lower and skeletal musclesodium channels were only minimally affected.

SUMMARY OF THE INVENTION

It has been an object of the present invention to provide compoundshaving a higher potency in blocking voltage operated sodium channelssuch as muscle sodium channels, neuronal sodium channels and cardiacsodium channels.

It has been a further object of the present invention to provide apharmaceutical composition, in particular a local anestheticcomposition, an anticonvulsive/antiepileptic composition, anantidysrhythmic composition and a spasmolytic composition which is moreeffective, i.e., has an improved potency.

The above objectives were solved by a pharmaceutical or cosmeticcomposition comprising a phenol derivative represented by formula (I)

wherein

-   -   R¹ represents a hydrogen atom, a halogen atom or hydrocarbon        group containing up to 12 carbon atoms;    -   R² represents a hydrogen atom or a C₁-C₇ alkyl group; wherein    -   R¹ and R² may optionally form a carbocyclic 5- or 6-membered        ring;    -   R³ represents a hydrogen atom, a halogen atom or a C₁-C₇ alkyl        group;    -   R⁴ represents a hydrogen atom, a C₁-C₇ alkyl group or a halogen        atom;    -   R⁵ represents a hydrogen atom, a halogen atom or a C₁-C₇ alkyl        group; and    -   R⁶ represents a hydrogen atom, a C₁-C₇ alkyl group or a C₂ or C₃        alkenyl group,    -   under the proviso that R² and R⁴ can only both represent a        hydrogen atom if both R¹ and R⁵ represent a C₁-C₇ alkyl group        and R³ represents a halogen atom.

The above composition has been found to have an improved potency inblocking sodium channels and/or influencing the kinetics of sodiumchannels in a mammal.

DESCRIPTION OF THE FIGURES

FIG. 1

Concentration-dependent reduction in test pulse current with respect tocontrol (I/I_(max), mean±SD) induced by the different compounds. Thedata were derived from at least four different experiments for eachconcentration tested. Depolarizing pulses to 0 mV (10 ms duration) werestarted from −150, −100, or −70 mV. Solid lines are Hill fits(I_(Na+)=[1+([C]/IC₅₀)^(nH)]⁻¹) to the data.

Concentration-response plots at −100 and −150 mV were nearlysuperimposable for all compounds, while the potency of the drugs wasmarkedly increased at −70 mV.

FIG. 2

Concentration-dependent reduction in test pulse current with (filledsquares) or without (empty triangles) a 2.5 s inactivating prepulseintroduced before the test pulse (n>4, mean±SD). Currents werenormalized to the current elicited with the same protocol in controlconditions. The solid lines are Hill fits to the data. The 2.5 sprepulse uniformly enhanced sensitivity to all compounds examined.

FIG. 3

Recovery from fast inactivation assessed by a two-pulse protocol incontrol conditions (circles) and in the presence of 100 μM3,5-dimethyl-4-chlorophenol (squares). The abscissa represents therecovery time interval between prepulse and test pulse (up to 100 ms),the ordinate represents the fractional current (mean±SD, n=5) afterrecovery from fast inactivation, induced by the prepulse in the sameseries. In the presence of drug, currents were normalized either to theprepulse in the presence of drug (filled symbols) or in thecorresponding control conditions (empty symbols). Solid lines areexponential fits I(t)=a₀+a₁exp(−t/τ_(h1))+a₂exp(−t/τ_(h2)) to thefractional currents after recovery from inactivation or inactivatedchannel block. Without drug, the data fitted to a monoexponential. Inthe presence of drug, recovery was delayed and contained a second slowcomponent of 94 ms, which made up 8% of the current amplitude.

DETAILED DESCRIPTION OF THE INVENTION

The hydrocarbon group containing up to 12 carbon atoms as defined inconjunction with substituent R¹ may be a straight or branched aliphaticor alicyclic group which may be substituted with at least onesubstituent selected from halogen, hydroxyl, and oxo groups (thusforming keto or aldehyde groups). Independently said hydrocarbon groupmay optionally contain at least one double-bond or said hydrocarbongroup may be an aromatic group (such as a phenyl group) which mayoptionally be substituted with at least one straight or branched C₁-C₆alkyl or C₂-C₆ alkenyl group. In a preferred embodiment said hydrocarbongroup contains up to 7 carbon atoms, more preferably 2 to 6 carbonatoms. Preferably, said hydrocarbon group may represent a C₁-C₇ alkylgroup.

Generally, in conjunction with the present invention the C₁-C₇ alkylgroup as defined for substituents R¹ to R⁶ denotes a branched orstraight (linear) chain hydrocarbon group which may optionally besubstituted with a halogen atom, a hydroxyl group or an oxo-group (═O).In another embodiment said hydrocarbon group contains 2 to 6, in a stillfurther embodiment up to 5 carbon atoms. Preferred alkyl-groups, whichmay be substituted or not as mentioned above, may be selected from thegroup consisting of methyl, ethyl, n-propyl, iso-propyl, n-butyl,iso-butyl, sec.-butyl, tent.-butyl, and the isomers of the pentyl andhexyl-group.

It is evident that R¹ and R² can only form a ring if R¹ is neither ahydrogen nor a halogen atom. The ring which may be formed by R¹ and R²may further contain at least one double bond.

In a preferred embodiment the 5- or 6-membered ring formed by R¹ and R²is a 5- or 6-membered saturated or unsaturated carbocyclic ring, towhich there can be annealed a phenyl ring, which may optionally besubstituted with a C₁-C₇ alkyl group.

In terms of the present invention the halogen atoms are selected fromfluorine, chlorine, bromine and iodine with chlorine and fluorine beingpreferred.

By a C₂-alkenyl group there is meant an ethenyl (CH₂═CH—) and by C₃alkylene group there is meant a propenyl (CH₂═CH—CH₂—) group.

The substituents R² and R⁴ can only both represent a hydrogen atom atthe same time if both R¹ and R⁵ represent a C₁-C₇ alkyl group and R³represents a halogen atom.

It is understood that alternative to the free phenols, i.e., if R⁶represents a hydrogen atom the respective alkalimetal phenolates, forinstance, the sodium phenolates, may also be employed. In a preferredembodiment in the above formula (I)

-   -   R¹ represents a hydrogen atom, a halogen atom or a C₁-C₇ alkyl        group;    -   R³ represents a hydrogen atom, a halogen atom or a C₁-C₇ alkyl        group;    -   R⁴ represents a C₁-C₇ alkyl group or a halogen atom; and    -   R⁵ represents a hydrogen atom, a halogen atom or a C₁-C₇ alkyl        group.

In an alternative embodiment in the above formula (I)

-   -   R¹ represents a hydrogen atom or a C₁-C₇ alkyl group;    -   R² represents a C₁-C₇ alkyl group; wherein    -   R¹ and R² may optionally form a carbocyclic 5- or 6-membered        ring;    -   R³ represents a halogen atom or a C₁-C₇ alkyl group;    -   R⁴ represents a C₁-C₇ alkyl group;    -   R⁵ represents a hydrogen atom or a C₁-C₇ alkyl group; and    -   R⁶ represents a hydrogen atom or a C₁-C₇ alkyl group.

In a still further embodiment in the above formula (I)

-   -   R¹ represents a hydrogen atom;    -   R² represents a C₁-C₇ alkyl group;    -   R³ represents a halogen atom;    -   R⁴ represents a C₁-C₇ alkyl group;    -   R⁵ represents a hydrogen atom; and    -   R⁶ represents a hydrogen atom.

Additionally, in the above formula (I)

-   -   R¹ and R⁵ may represent a C₁-C₇ alkyl group, preferably a C₁-C₅        alkyl group, more preferably an iso-propyl-group;    -   R² and R⁴ may represent a hydrogen atom;    -   R³ may represent a halogen atom, preferably a chlorine or a        bromine atom; and    -   R⁶ may represent a hydrogen atom or a C₁-C₇ alkyl group.

Caused by the high blocking potency in combination with interferencewith channel gating in voltage-gated sodium channels the compounds canbe applied in low dosages as antidisrhythmic, spasmolytic,anticonvulsant/antiepileptic and local anesthetic.

The compounds according to the present invention have a higher potencyin sodium channel blockade compared to conventional, state of the artcompounds. For example, the IC₅₀ value for lidocaine block ofheterologously expressed muscle sodium channels at −100 mV was 500 μM(Fan et al., J. Physiol., (1996) 275-286), compared with 150 μM for3,5-dimethyl-4-chlorophenol, i.e., a compound according to the presentinvention. It has surprisingly been found that the blocking potency ofphenol derivatives is increased by halogenation and by increasing thenumber of alkyl groups. In addition, it has been found thatvoltage-dependent block by all compounds retains a characteristic set offeatures that describes local anesthetic block.

Exemplary compounds which can be used according to the present inventionare the substituted phenols (II) to (VI).

In experiments it was found that all substituted phenols blockedskeletal muscle sodium channels in a concentration-dependent manner.Compounds which are not according to the invention such as3-methylphenol and 4-chlorophenol blocked sodium currents ondepolarization from -100 mV to 0 mV with IC₅₀ values of 2161 μM and 666μM, respectively. Methylation of the halogenated compound furtherincreased potency, reducing the IC₅₀ to 268 μM in4-chloro-2-methylphenol. The IC₅₀ was surprisingly reduced further to150 μM in 3,5-dimethyl-4-chlorophenol, a compound falling under thescope of formula (I).

Membrane depolarization before the test depolarization significantlypromoted sodium channel blockade. When depolarizations were started from−70 mV or when a 2.5 s prepulse was introduced before the test pulseinducing slow inactivation, the IC₅₀ was reduced more than three-fold byall compounds. The values of K_(D) for the fast-inactivated statederived from drug-induced shifts in steady-state availability curveswere 14 μM for 3,5-dimethyl-4-chlorophenol. For comparative compoundsthere was found 19 μM for 4-chloro-2-methylphenol, 26 μM for4-chlorophenol and 115 μM for 3-methylphenol.

All compounds used according to the invention accelerated the currentdecay during depolarization and slowed recovery from fast inactivation.No relevant frequency-dependent block after depolarizing pulses appliedat 10, 50, and 100 Hz was detected for any of the compounds.

All the phenol derivatives according to the present invention areeffective blockers of sodium channels, such as skeletal muscle sodiumchannels, especially in conditions that are associated with membranedepolarization.

Typically, the pharmaceutical compositions according to the presentinvention can be applied in various forms such as in the form ofemulsions (oil-in-water), sprays, ointments, creams, pastes andcapsules. The pharmaceutical compositions thus may be in injectable formor in topically applicable form.

Injection solutions for parenteral administration comprise between about0.1 and about 5% by weight, preferably about 0.2 to about 3% by weightand more preferably up to about 2% by weight of the phenol derivative offormula (I). For infiltrative anesthesia the solutions contain betweenabout 0.4 and about 0.7% by weight of the active ingredient. Forconduction anesthesia the solutions contain between about 1 and about 2%by weight, for epidural anesthesia between about 0.5 and about 1% byweight of the active ingredient. For local topical anesthesia the phenolderivative of formula (I) is contained in the ointment, cream or pastein an amount of about 0.5 to about 5% by weight, preferably in an amountof about 1 to about 3% by weight, typically in an amount of up to about2% by weight.

The manufacture of parenteral solutions is conventional in the art. Dueto the increased potency of the phenol derivatives the dosage isdecreased significantly.

While the invention has been described in connection with a number ofpreferred embodiments thereof, those skilled in the art will recognizethat many modifications and changes can be made therein withoutdeparting from the scope of the invention.

The present invention is further described by the experiments whichfollow hereinafter. The scope of the invention is not limited to theseexperiments.

EXPERIMENTS

The sodium channel blocking potency effects of different phenolderivatives with methyl and halogen substituents have been studied onheterologously expressed human skeletal muscle sodium channels. Thephenol derivatives encompassed by formula (I) are either commerciallyavailable or can be synthesized by conventional standard laboratorymethods.

Molecular Biology

Wild type α-subunits of human muscle sodium channels were heterologouslyexpressed in human embryonic kidney (HEK 293) cells, a stable cell linesince 1962 (American Tissue Culture Collection CRL 1573). Transfectionwas performed using calcium phosphate precipitation (Graham & Van derEb, Virology, 52 (1973) 456-467). Permanent expression was achieved byselection for resistance to the aminoglycoside antibiotic geneticin G418(Life Technology, Eggenstein, Germany) (Mitotic et al., J. Physiol., 478(1994) 395-402). Successful channel expression was verifiedelectrophysiologically. The clone has been used in severalinvestigations (Haeseler et al.,Br. J. Pharmacol., 128 (1999) 1259-67;Haeseler et al., Br. J. Pharmacol. 130 (2000) 1321-1330; Mitrovic etal., ibid.).

Solutions

3,5-dimethyl-4-chlorophenol (according to the invention) and3-methylphenol (not according to the invention) were purchased fromSigma Chemicals, Deisenhofen, Germany; 4-chloro-2-methylphenol (notaccording to the invention) and 4-chlorophenol (not according to theinvention) were from FLUKA, Deisenhofen, Germany.3,5-Dimethyl-4-chlorophenol was prepared as a 1 M stock solution inmethanol; 4-chloro-2-methylphenol, 4-chlorophenol, and 3-methylphenolwere dissolved directly in the bath solution immediately before theexperiments. Concentrations were calculated from the amount injectedinto the glass vials. Drug-containing solutions were protected fromlight and were vigorously vortexed for 60 min. The solution was appliedvia a glass polytetrafluoroethylene perfusion system and a stainlesssteel superfusion pipette. The bath solution contained (mM) NaCl 140,MgCl₂ 1.0, KCl 4.0, CaCl₂ 2.0, Hepes 5.0, dextrose 5.0. Patch electrodescontained (mM) CsCl₂ 130, MgCl₂ 2.0, EGTA 5.0, Hepes 10. All solutionswere adjusted to 290 mosm/l by the addition of mannitol and to pH 7.4 bythe addition of CsOH.

Experimental Set-Up

Standard whole-cell voltage-clamp experiments (Hamill et al., PfuegersArch., 391 (1981) 85-100) were performed at 20° C. Each experimentconsisted of test recordings with the drug present at only oneconcentration, and of drug-free control recordings before and after thetest. Each whole-cell patch was exposed to one test concentration only.At least four experiments were performed at each concentration. Theamount of the diluent methanol corresponding to the test concentrationof 3,5-dimethyl-4-chlorophenol was added to the control solution.Patched cells were lifted into the visible stream of either bathsolution or test solution, applied via a two-channel superfusion pipetteclose to the cell. To ensure adequate adjustment of the applicationdevice, one test experiment in distilled water reducing inward sodiumcurrent to zero was performed every 6-10 experiments.

Current Recordings and Analysis

For data acquisition and further analysis we used the EPC9digitally-controlled amplifier in combination with Pulse and Pulse Fitsoftware (HEKA Electronics, Lambrecht, Germany). The EPC9 providesautomatic subtraction of capacitive and leakage currents by means of aprepulse protocol. The data were filtered at 10 kHz and digitized at 20μs per point. Input resistance of the patch pipettes was at 1.8-2.5 MΩ.Only small cells with capacitances of 9-15 pF were used; residual seriesresistance (after 50% compensation) was 1.2-2.5 MΩ; experiments with arise in series resistance were rejected. The time constant of thevoltage settling within the membrane (residual series resistance×cellcapacitance) was less than 35 μs. To minimize a possible contribution ofendogenous Na⁺ channels in HEK cells that conduct with amplitudesranging from 50 to 350 pA (Mitrovic et al., 1994, ibid.), but also toavoid large series resistance errors, only currents ranging between 1and 6 nA were analysed. To minimize time-dependent shifts in thevoltage-dependence of steady-state inactivation (Wang et al., 1996), alltest experiments were performed within 5 min of patch rupture. Underthese experimental conditions, time-dependent hyperpolarizing shifts incontrol conditions were less than −2 mV (Haeseler et al., Anesthiology,92 (2000) 1385-92). Voltage-activated currents were studied by applyingdifferent voltage-clamp protocols. Either exponential functions[I(t)=a₀+a₁ exp(−t/τ_(h1))+a₂ exp(−t/τ_(h2))] or Boltzmann functions[I/I_(max)=(1+exp(−zF(V_(test)−V_(0.5))/RT))⁻¹] were fitted to the data,using a non-linear least-squares Marquardt-Levenberg algorithm, yieldingthe time constant τ of inactivation and recovery from inactivation, themembrane potential at half-maximum channel availability (V_(0.5)), andthe slope factor z of the steady-state availability curve. F isFaraday's constant (9.6487×10⁴ C mol⁻¹), R is the gas constant (8.315 JK⁻¹ mol⁻¹), and T is the temperature in degrees Kelvin. Drug effects onthe peak current amplitude were assessed at different holding potentials(−70, −100 and −150 mV), or when a 2.5 s prepulse to −35 mV wasintroduced before the test pulse in order to induce slow inactivation.All data are presented as mean ±SD. The residual sodium current(I_(Na+)) in the presence of drug (with respect to the current amplitudein control solution) was plotted against the applied concentration ofeach drug [C]. Fits of the Hill equation [I_(Na+)=(1+([C]/IC₅₀)^(n) ^(H))⁻¹] to the data yielded the concentration for half-maximum channelblockade (IC₅₀) and the Hill coefficient n_(H).

Results

Successful Na⁺ channel expression was verified electrophysiogically inalmost all of the established whole-cell patches. In all 83 cells wereincluded in the study. Average currents in the control experiments afterdepolarization from −100 mV to 0 mV were 4.9±2.1 nA.

Suppression of Peak Sodium Currents-Differences in Potency Related toMethylation and/or Halogenation of the Phenol Ring

Maximum inward currents elicited by 10 ms pulses going from either −150mV, −100 mV, or −70 mV to 0 mV were reversibly suppressed by allsubstances in a concentration-dependent manner. Suppression occurredwithin 60 s after the start of perfusion with the drug-containingsolution. The currents in the presence of drug were normalized to therespective current elicited in control conditions. Normalized currentsderived from at least four different experiments for each drugconcentration were averaged to establish concentration-response plots(see FIG. 1).

The degree of suppression at all holding potentials increased withhalogenation and with the number of methyl groups at the phenol ring.The phenol derivative 3-methylphenol, containing only one methyl groupin the meta position with respect to the hydroxyl group, blocked inwardsodium current at a holding potential of −150 mV, with an IC₅₀ value of2395 μM.

The halogenated compound 4-chlorophenol was more potent than themethylated compound, and reduced the IC₅₀ to 751 μM. Methylation inaddition to halogenation further increased potency, reducing the IC₅₀about two-fold for each methyl group inserted into the halogenatedcompound (316 μM for 4-chloro-2-methylphenol and 162 μM for3,5-dimethyl-4-chlorophenol).

Acceleration of the Na⁺ Current Decay Phase by Phenol Derivatives

To examine the time course of Na⁺ channel inactivation during adepolarization, 40 ms voltage steps from a holding potential of −100 mVto 0 mV were performed. The time constant of channel inactivation τ_(h)was obtained by fitting a single exponential to the decay of currentduring depolarizations: I(t)=a₀+a₁ exp(−t/τ_(h)). In control conditions,τ_(h) was 0.43±0.08 ms (n=72). All phenol derivatives accelerated thedecay of whole-cell currents. For all compounds, however, this effectwas apparent only at concentrations that exceeded the IC₅₀ values at aholding potential of −70 mV. Values obtained for τ_(h) in the presenceof drug were: 0.27±0.02 ms in 50 μM 3,5-dimethyl-4-chlorophenol,0.27±0.05 ms in 100 μM 4-chloro-2-methylphenol, 0.23±0.03 ms in 500 μM4-chlorophenol, and 0.30±0.08 ms in 1000 μM 3-methylphenol.

Effects of Phenol Derivatives on Recovery from Fast Inactivation

After inactivation, channel re-openings are impossible until thechannels recover from inactivation, a process that requires several msafter membrane repolarization. Further information about drug effects onthe stability of the fast-inactivated state or the kinetics of drugdissociation from the fast-inactivated state can be derived from therate at which the channels recover from inactivation in the presence ofthe drug. The time of membrane repolarization required to remove fastinactivation was assessed at −100 mV by a two-pulse protocol withvarying time intervals (up to 100 ms) between the inactivating prepulseand the test pulse (see FIG. 3). The time constants of recovery,τ_(rec), were derived from monoexponential or biexponential fits to thefractional current after recovery from inactivation, plotted against thetime interval between the inactivating prepulse and the test pulse:I(t)=a₀+a₁ exp(−t/τ_(rec1))+a₂ exp(−t/τ_(rec2)) Without drug, the datafitted well to a monoexponential, yielding a time constant, τ_(rec1), of2.3±0.7 ms (n=34). In the presence of drug, the fit contained a second,slow component of recovery, τ_(rec2) of 94±8 ms(3,5-dimethyl-4-chlorophenol), 36±5 ms (4-chloro-2-methylphenol), and30±0.1 ms 4-chlorophenol and 3-methylphenol). For all drugs, however,the slow component made up less than 10% of the current amplitude atconcentrations close to the IC₅₀ for rest block. The fast component,τ_(rec1), was prolonged to 3.8±0.8 ms in 100 μM3,5-dimethyl-4-chlorophenol, to 4.3±1.6 ms in 300 μM4-chloro-2-methylphenol, to 3.0±0.7 ms in 500 μM 4-chlorophenol, and to2.8±0.6 ms in 1000 μM 3-methylphenol. FIG. 3 shows the time-course ofrecovery from fast inactivation with and without 100 μM3,5-dimethyl-4-chlorophenol.

Frequency-Dependent Block

The accumulation of block during trains of depolarizing pulses indicatesthat the interval between pulses is too short to allow recovery of Na⁺channel availability. To derive an estimate of the kinetics of drugbinding and unbinding during the interpulse interval, we applied seriesof 1-10 ms depolarizing pulses from −100 mV to 0 mV at high frequencies(10, 50, and 100 Hz).

Frequency-dependent block was defined as the additional reduction inI_(Na+) for the last pulse relative to the first pulse in a test trainin the presence of drug.

In control conditions, the amplitude of the last pulse relative to thefirst pulse in a test train was 99±1% at 10 Hz and 96±3% at 50 and 100Hz. Neither compound induced frequency-dependent block over 10% at 10Hz. At 50 and 100 Hz, only concentrations exceeding the IC₅₀ for restblock produced a small amount of frequency-dependent block. During a 100Hz train, the additional fall relative to the first pulse was 17±5% in300 μM 3,5-dimethyl-4-chlorophenol, 16±5% in 500 μM4-chloro-2-methylphenol, 13±2% in 1000 μM 4-chlorophenol, and 8±2% in3000 μM 3-methylphenol.

1. The method for treatment of a human or animal body which comprisesadministering an effective amount of a phenol derivative represented byformula (VI) for the blockade of sodium channels and/or for influencingthe kinetics of sodium channels:


2. The method for treatment of a human or animal body which comprisesadministering an effective amount of a phenol derivative represented byformula (I) for the blockade of sodium channels and/or for influencingthe kinetics of sodium channels:

Wherein, R¹ and R⁵ each represents a C₂₋C₆ alkyl group; R², R⁴, and R⁶each represents a hydrogen atom, and R³ represents a halogen atom. 3.The method of claim 2 wherein R³ is an iodine atom.
 4. The method ofclaim 2 wherein R³ is a chlorine or bromine atom.
 5. The method of claim2 wherein an effective amount of a phenol derivative of formula (I) isadministered for the treatment of dysrhyhthmia.